The goal of our research is to understand the mechanism of cyclooxygenase catalysis by PGH synthases (PGHSs). PGHS-1 and -2 catalyze the formation of prostaglandin endoperoxide H2 (PGH/2) from arachidonic acid--the committed step in prostaglandin biosynthesis. Both enzymes catalyze (a) a cyclooxygenase reaction in which arachidonate is converted to PGH/2 and (b) a peroxidase reaction in which PGH/2 is reduced to PGH/2. These two reactions occur at distinct but interconnected cyclooxygenase and peroxidase sites that are bisected by a heme group. The cyclooxygenase active site is a hydrophobic tunnel that protrudes from the membrane binding surface of PGHS into the major globular domain of the enzyme. Fatty acid substrates and probably 02 enter this site from the membrane at the base of the tunnel. The peroxidase site is located on the opposite surface of the protein and resembles that of myeloperoxidase. Our first specific aim to characterize the contributions of amino acids within the cyclooxygenase active site to the binding of arachidonic acid. We will determine the crystal structure of a cyclooxygenase-inactive Y371F human (h) PGHS-2 mutant with arachidonate bound in the active site. We will also prepare mutations of various active site amino acids and analyze the effects of these changes on substrate binding and catalysis. Our second specific aim is to determine if suicide inactivation of cyclooxygenase activity results from radical-initiated intramolecular protein cross linking involving a Tyr504 radical. We will characterize peptide products derived from native and suicide-inactivated ovine PGHS-1 to determine what residues are modified during inactivation. We will also determine the rates of formation of various spectral intermediates formed upon interaction of hydroperoxides with H386A ovine PGHS-1, a mutant PGHS that fails to undergo inactivation. Finally, we will determine the effect f replacing Tyr504. Our third specific aim is to determine if the H20 channel of PGHS that connects the cyclooxygenase active site with the exterior of the protein serves as a conduit for the proton that is abstracted from the active site Tyr385 during cyclooxygenase catalysis; we will characterize mutations likely to block the H20 channel (G227A, G536A) and mutations expected to negate proton transfer (R37G,L).